HIV envelope V3-CCR5 binding site immunogen

ABSTRACT

The present invention relates, in general, to an immunogen and, in particular, to an immunogen for inducing antibodies that neutralize a wide spectrum of HIV primary isolates. The invention also relates to a method of inducing anti-HIV antibodies using same.

[0001] This application claims priority from U.S. ProvisionalApplication No. 60/333,148, filed Nov. 27, 2001, the entire content ofwhich is incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates, in general, to an immunogen and,in particular, to an immunogen for inducing antibodies that neutralize awide spectrum of HIV primary isolates. The invention also relates to amethod of inducing anti-HIV antibodies using such an immunogen.

BACKGROUND

[0003] As the HIV epidemic continues to spread world-wide, the need foran effective HIV vaccine remains urgent. A key obstacle to HIV vaccinedevelopment is the extraordinary variability of HIV and the rapidity andextent of HIV mutation (Wain-Hobson in The Evolutionary biology ofRetroviruses, SSB Morse Ed. Raven Press, NY, pgs 185-209 (1994))

[0004] Myers, Korber and colleagues have analyzed HIV sequencesworldwide and divided HIV isolates into groups or clades, and provided abasis for evaluating the evolutionary relationship of individual HIVisolates to each other (Myers et al (Eds), Human Retroviruses and AIDS(1995), Published by Theoretical Biology and Biophysics Group, T-10,Mail Stop K710, Los Alamos National Laboratory, Los Alamos, N. Mex.87545). The degree of variation in HIV protein regions that contain CTLand T helper epitopes has also recently been analyzed by Korber et al,and sequence variation documented in many CTL and T helper epitopesamong HIV isolates (Korber et al (Eds), HIV Molecular ImmunologyDatabase (1995), Published by Theoretical Biology and Biophysics Group,Los Alamos National Laboratory, Los Alamos, N. Mex. 87545).

[0005] A new level of HIV variation complexity was recently reported byHahn et al by demonstrating the frequent recombination of HIV amongclades (Robinson et al, J. Mol. Evol. 40:245-259 (1995)). These authorssuggest that as many as 10% of HIV isolates are mosaics ofrecombination, suggesting that vaccines based on only one HIV clade willnot protect immunized subjects from mosaic HIV isolates (Robinson et al,J. Mol. Evol 40:245-259 (1995)).

[0006] The present invention relates to an immunogen suitable for use inan HIV vaccine. The immunogen will induce broadly cross-reactiveneutralizing antibodies in humans and neutralize a wide spectrum of HIVprimary isolates.

SUMMARY OF THE INVENTION

[0007] The present invention relates to an immunogen for inducingantibodies that neutralize a wide spectrum of HIV primary isolates. Theinvention also relates to a method of inducing anti-HIV antibodies usingsuch an immunogen.

[0008] Objects and advantages of the present invention will be clearfrom the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1. Peptide immunogen design.

[0010]FIG. 2. Sequence of CBLH-1-89.6P.

DETAILED DESCRIPTION OF THE INVENTION

[0011] Targets that induce antibodies that neutralize primary isolatesof HIV include the gp120V3 loop and the CCR5 (cellular HIV co-receptor)binding site. Kwong et al (Nature 393:648-659 (1968)) have shown thatthe CCR5 binding site is adjacent to the base of the V3 loop and isformed by the juxtaposition of 4 anti-parallel beta-pleated sheets. Thepresent invention provides a peptide immunogen that induces antibodiesthat neutralize HIV primary isolates comprising components of both theHIV gp120 CCR5 binding site and the V3 loop. The immunogen has thedesign set forth in FIG. 1.

[0012] The peptide immunogen of the invention, designated CCR5 bindingsite/V3, with the CBLH-1 peptide being the prototype, comprises, fromthe N-terminus to the C-terminus, beta sheet regions 20, 21, 2 and 3(see Nature 393:650 (1998)). A V3 loop sequence connects beta sheets 21and 2 and a V3 loop sequence is present between beta sheets 2 and 3,which site is naturally occupied by the V1-V2 loops. Accordingly, thepeptide immunogen of the invention comprises 4 anti-paralleled betasheet sequences that reflect the CCR5 binding site and 2 V3 loops. TheV3 loops can vary in length (for example, from about 8 to about 16 aminoacids). In a preferred embodiment, the 4 beta sheets correspond todisparate gp120 regions. In CCR5 binding site/V3, CBLH-1, they arepresent in a linear peptide comprising V3 loops.

[0013] A multiplicity of peptide immunogens of the present invention canbe formulated as a composition suitable for administration as a vaccine.The V3 components of the peptide immunogens of the invention present inthe instant composition are selected so as to be representative ofhigher order structural motifs present in a population, which motifsmediate V3 functions in the course of envelope mediated HIV interactionwith host cells. The Los Alamos National Laboratories Human Retrovirusesand AIDS Database (Human Retroviruses and AIDS, 2000, Published by theTheoretical Biology and Biophysics G T-10, Mail Stop K710, LANL, LosAlamos, N. Mex.) presently contains over 14,000 HIV V3 envelopesequences, showing the extraordinary diversity the virus has obtainedsince originating in man in Africa approximately 50 years ago. Forexample, among 432 HIV-1 V3 sequences derived from individuals infectedwith subtype C (designated “Clade C”) in Africa currently available inthe HIV database, 176 distinct variants of a 23 amino acid stretch atthe tip of the V3 loop have been found. Similarly, among 6870 B subtype(designated “Clade B”) V3 sequences from the US, 1514 unique forms havebeen found.

[0014] A method has been developed to organize short antigenic domainsby protein similarity scores using maximum-linkage clustering. Thismethod enables the visualization of the clustering patterns as adendrogram, and the splitting patterns in the dendrogram can be used todefine clusters of related sequences (Korber et al, J. Virol.68:6730-6744 (1994)). The method allows the use of several differentamino acid similarity scoring schemes available in the literature,preferred is the amino acid substitution matrix developed by Henikoffand Henikoff (see Advances in Protein Chemistry 54:73-97 (2000) andProteins: Structure, Function and Genetics 17:49-61 (1993)), designed togive substitutions that are well tolerated in conserved proteinstructural elements a high score, and a low score to those that are not.Typically excluded from consideration very rare, highly divergentpeptides, and favored are peptides found in many individuals within thepopulation. In a selected set of sequences, most of the unique forms arewithin one or two amino acids from a least one other of the peptideschosen. This method has been applied to clustering the large number ofvariants of the antigenic tip of the V3 domain within Clade B and CladeC into groups (about 25) that are likely to be cross-reactive within thegroup. Based on these clustering patterns, variants (e.g., about 25-30)are selected that are representative or “central” to each group, fortesting for antigenicity. The HIV Clade B and Clade C gp120 envelope V3sequences have been analyzed, as described above, for groups of V3sequences predicted to have structural similarities. Twenty five Clade Cand 30 Clade B groups have been defined, and chosen out of each group isa common, or the most common, sequence as a representative of thatgroup.

[0015] Shown in Tables 3 and 4 are examples of immunogens of the presentinvention for HIV Clades B and C, respectively. The immunogens of B canbe combined to provide a composition suitable for use in the US (cladeB) and Africa (Clade C). TABLE 3396.2/170.6-RIKQIINMWQKVGKAMYA-RRNIHIGLGRRF-SLKPCVKTPLCV-RRSVRIGPGGAM-SCNTSVITQA82.15/144.8-RIKQIINMWQKVGKAMYA-RRSIPIGPGRAF-SLKPCVKTPLCV-VRKIPIGPGSSF-SCNTSVITQA23.38/365.2-RIKQIINMWQKVGKAMYA-RKRIPLGLGKAF-SLKPCVKTPLCV-RKGIHLGPGPAI-SCNTSVITQA513.2/1448.1-RIKQIINMWQKVGKAMYA-RKGIHMGPGKAI-SLKPCVKTPLCV-RRGIPIGPGRAF-SCNTSVITQA69.18/146.8-RIKQIINMWQKVGKAMYA-RKSIRIGPGRAV-SLKPCVKTPLCV-RRRISIGPGRAF-SCNTSVITQA113.10/51.23-RIKQIINMWQKVGKAMYA-RRSIHLGMGPAL-SLKPCVKTPLCV-RRSIHMGLGRAF-SCNTSVITQA72.18/36.29-RIKQIINMWQKVGKAMYA-RKGINIGPGRAF-SLKPCVKTPLCV-RKGIHIGPGRTF-SCNTSVITQA70.18/89.14-RIKQIINMWQKVGKAMYA-IRIGHIGPGRAF- SLKPCVKTPLCV-RRHIHIGPGRAF-SCNTSVITQA 163.7/57.20-RIKQIINMWQKVGKAMYA-RRKGIHIGPGRAI-SLKPCVKTPLCV-TGKSIRMGLGRAW-SCNTSVITQA11.85/34.29-RIKQIINMWQKVGKAMYA-RKSINIGPGRAF-SLKPCVKTPLCV-RKSIQIGPGPAF-SCNTSVITQA1.481/85.15-RIKQIINMWQKVGKAMYA-RKSIHIGPGRAF-SLKPCVKTPLCV-RKSIHIAPGRAF-SCNTSVITQA62.19/125.9-IKQIINMWQKVGKAMYA-RKSIHIGPGRAF-SLKPCVKTPLCV-RRRISMGPGRVL-SCNTSVITQA35.29/74.17-RIKQIINMWQKVGKAMYA-RKRISLGPGRVY-SLKPCVKTPLCV-RKRMTLGPGKVF-SCNTSVITQA46.26/122.9-RIKQIINMWQKVGKAMYA-QRIIHIGPGRPF-SLKPCVKTPLCV-RIRIHRGYGRSF-SCNTSVITQA162.7/3.323-RIKQIINMWQKVGKAMYA-RGSIHLHPGRKF-SLKPCVKTPLCV-RKSINMGPGRAF-SCNTSVITQA

[0016] TABLE 4 1(4)-RIKQIINMWQKVGKAMYA-rksirigpGqtf-SLKPCVKTPLCV-rksVrigpGqtf-SCNTSVITQA7(8)-RIKQIINMWQKVGKAMYA-rEsirigpGqtf-SLKPCVKTPLCV-rRsorogpGqAF-SCNTSVITQA9(10)-RIKQIINMWQKVGKAMYA-rkGirigpGqtf-SLKPCVKTPLCV-rksirigpGqAF-SCNTSVITQA14(15)-RIKQIINMWQKVGKAMYA-rksMrigpGqtf-SLKPCVKTPLCV-rksirigpGqtL-SCNTSVITQA16(17)-RIKQIINMWQKVGKAMYA-rksVrigpGqtS-SLKPCVKTPLCV-rRsirigpGqtf-SCNTSVITQA20(22)-RIKQIINMWQKVGKAMYA-rQsirigpGqAF-SLKPCVKTPLCV-rksVrigpGqAF-SCNTSVITQA23(24)-RIKQIINMWQKVGKAMYA-rkGiHigpGqAf-SLKPCVKTPLCV-rkGiGigpGqtf-SCNTSVITQA25(14)-RIKQIINMWQKVGKAMYA-rEsiGigpGqAf-SLKPCVKTPLCV-rksMrigpGqtf-SCNTSVITQA

[0017] While the above is offered by way of example, it will beappreciated that the same analyses can by performed for HIV Clades A, D,E, F, G, H, M, N, O, etc, to design immunogens that react with HIVprimary isolates from these Clades. The length of the V3 inserts in thepresent immunogens can vary, for example, from about 8 to about 16 aminoacids. In a similar manner, analysis can be made of amino acidheterogeneity with the 2, 3, 20 and 21 beta sheet regions of gp120 andmultiple HIV (chemokine) receptor binding site sequences can be used inpeptide design.

[0018] The peptide immunogens of the invention can be chemicallysynthesized and purified using methods which are well known to theordinarily skilled artisan. The composition can comprise the peptideslinked end to end or can comprise a mixture of individual peptides. Thepeptide immunogens can also be synthesized by well-known recombinant DNAtechniques. Recombinant synthesis may be preferred when the peptides arecovalently linked.

[0019] Nucleic acids encoding the peptides of the invention can be usedas components of a DNA vaccine wherein the peptide encoding sequence(s)is/are administered as naked DNA or, for example, a minigene encodingthe peptides can be present in a viral vector, such as an adenoviralvector, a modified vaccinia ankara vector, a vaccinia vector or anattenuated TB vector. Expression of the immunogenic peptides of theinvention can be induced in a patient's own cells, by introduction intothose cells of nucleic acids that encode the peptides, preferably usingcodons and promoters that optimize expression in human cells. Examplesof methods of making and using DNA vaccines are disclosed in U.S. Pat.Nos. 5,580,859, 5,589,466, and 5,703,055.

[0020] The composition of the invention comprises an immunologicallyeffective amount of the peptide immunogens of this invention, or DNAsequence(s) encoding same, in a pharmaceutically acceptable deliverysystem. The compositions can be used for prevention and/or treatment ofimmunodeficiency virus infection. The compositions of the invention canbe formulated using adjuvants, emulsifiers, pharmaceutically-acceptablecarriers or other ingredients routinely provided in vaccinecompositions. Optimum formulations can be readily designed by one ofordinary skill in the art and can include formulations for immediaterelease and/or for sustained release, and for induction of systemicimmunity and/or induction of localized mucosal immunity (e.g, theformulation can be designed for intranasal administration). The presentcompositions can be administered by any convenient route includingsubcutaneous, intranasal, oral, intramuscular, or other parenteral orenteral route. The immunogens can be administered as a single dose ormultiple doses. Optimum immunization schedules can be readily determinedby the ordinarily skilled artisan and can vary with the patient, thecomposition and the effect sought. By way of example, it is noted thatapproximately 50 μg-100 μg of each hybrid peptide can be administered,for example, intramuscularly (e.g. 3×).

[0021] The invention contemplates the direct use of both the peptides ofthe invention and nucleic acids encoding same. For example, a minigeneencoding the peptides can be used as a prime and/or boost.

[0022] In addition to the composition described above, the inventionencompasses each of the immunogens disclosed as well as each of thecomponents (V3 and CCR5), alone or in covalent or non-covalentassociation with other sequences. The invention further encompassesnucleic acid sequences encoding any and all such peptides.

[0023] Certain aspects of the invention are described in greater detailin the non-limiting Example that follows.

EXAMPLE

[0024] A peptide immunogen of the invention, designated CBLH-1-89.6P)and having the sequence shown in FIG. 2 was tested for bothimmunogenicity with antibodies against the peptide and for neutralizingantibodies. Shown in Table 1 are the results of immunization of guineapigs twice with CBLH-1 of SHIV89.6P in complete Freund's adjuvant(CFA)/incomplete Freund's adjuvant (IFA) versus immunization of guineapigs twice with another immunogen, the C4-V3 gp120 immunogen (seeProvisional Application No. 60/331,036). TABLE 1 Titer to ImmunizingAnimal peptide after number Immunogen 2 Immunizations 322 CBLH-1 ofSHIV89.6P 102,400 323 CBLH-1 of SHIV 89.6P 204,800 324 CBLH-1 of SHIV89.6P 102,400 325 C4-V3 89.6P 25,600 326 C4-V3 89.6P 12,800 327 C4-V389.6P 12,800

[0025] Table 2 shows the neutralizing antibody results of the sera ofthe same animals against several HIV primary isolates. TABLE 2 Nab titer% p24 reduction Immun- in MT-2 cells¹ in PBMC² Animal ogen Bleed HIV-1MNSHIV-89.6P SF162 JR-FL 322 CBLH-1 Pre 0 0 0 0 1 0 0 0 0 2 0 0 0 0 3 1220 0 0 4 75 0 0 0 323 CBLH-1 Pre 0 0 0 0 1 42 0 0 0 2 444 0 0 0 3 >540 0100 88 4 >540 0 100 0 324 CBLH-1 Pre 0 0 0 0 1 0 0 0 0 2 188 0 0 03 >540 0 89 0 4 >540 24 93 0 325 C4-V3 Pre 0 0 0 0 89.6P 1 0 0 0 0 2 0 00 0 3 0 0 0 0 4 0 23 0 0 326 C4-V3 Pre 0 0 0 0 89.6P 1 79 0 0 0 2 131 00 0 3 53 0 0 0 4 47 0 0 0 327 C4-V3 Pre 0 0 0 0 89.6P 1 81 0 0 0 2 0 0 03 0 0 0 4 0 0 0

[0026] The results shown in Table 2 demonstrate that whereas C4-V3neutralization titers were low and did not cross neutralize any HIVprimary isolates, CBLH-1 of SHIV89.6P immunization of animals 323 and342 induced antibodies that cross-neutralized HIV SF162 and animal 323also cross-neutralized the primary isolate HTV JR-FL.

[0027] The following peptides have also been designed and may representimmunogenic truncated variants of CCR5 binding site/V3 peptideconstructs:

[0028] 1.481/85.15-Delta 20/21-RKSIHIGPGRAF-SLKPCVKTPLCV-RKSIHIAPGRAF-SCNTSVITQA

[0029] 1.481/85.15-Delta 2-RIKQIINMWQKVGKAMYA-RKSIHIGPGRAF-RKSIHIAPGRAF-SCNTSVTTQA

[0030] 1.481/85.15-Delta 2/3-RIKQIINMWQKVGKAMYA-RKSIHIGPGRAF-RKSIHIAPGRAF

[0031] 1.481/85.15-Delta 3-RTKQIINMWQKVGKAMYA-RKSIHIGPGRAF-SLKPCVKTPLCV-RKSIHIAPGRAF

[0032] 1.481/85.15-Delta 20/21/3-RKSIHIGPGRAF-SLKPCVKTPLCV-RKSIHIAPGRAF.

[0033] All documents cited above are hereby incorporated in theirentirety by reference.

[0034] One skilled in the art will appreciate from a reading of thisdisclosure that various changes in form and detail can be made withoutdeparting from the true scope of the invention.

1 29 1 64 PRT Artificial Sequence Description of Artificial SequenceHuman Immunodeficiency Virus 1 Arg Ile Lys Gln Ile Ile Asn Met Trp GlnLys Val Gly Lys Ala Met 1 5 10 15 Tyr Ala Arg Lys Ser Ile Asn Ile GlyPro Gly Arg Ala Phe Ser Leu 20 25 30 Lys Pro Cys Val Lys Thr Pro Leu CysVal Arg Lys Ser Ile Gln Ile 35 40 45 Gly Pro Gly Arg Ala Phe Ser Cys AsnThr Ser Val Ile Thr Gln Ala 50 55 60 2 64 PRT Artificial SequenceDescription of Artificial Sequence Human Immunodeficiency Virus 2 ArgIle Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met 1 5 10 15Tyr Ala Arg Lys Ser Ile His Ile Gly Pro Gly Arg Ala Phe Ser Leu 20 25 30Lys Pro Cys Val Lys Thr Pro Leu Cys Val Arg Lys Ser Ile His Ile 35 40 45Ala Pro Gly Arg Ala Phe Ser Cys Asn Thr Ser Val Ile Thr Gln Ala 50 55 603 63 PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 3 Ile Lys Gln Ile Ile Asn Met Trp Gln Lys Val GlyLys Ala Met Tyr 1 5 10 15 Ala Arg Lys Ser Ile His Ile Gly Pro Gly ArgAla Phe Ser Leu Lys 20 25 30 Pro Cys Val Lys Thr Pro Leu Cys Val Arg ArgArg Ile Ser Met Gly 35 40 45 Pro Gly Arg Val Leu Ser Cys Asn Thr Ser ValIle Thr Gln Ala 50 55 60 4 64 PRT Artificial Sequence Description ofArtificial Sequence Human Immunodeficiency Virus 4 Arg Ile Lys Gln IleIle Asn Met Trp Gln Lys Val Gly Lys Ala Met 1 5 10 15 Tyr Ala Arg LysArg Ile Ser Leu Gly Pro Gly Arg Val Tyr Ser Leu 20 25 30 Lys Pro Cys ValLys Thr Pro Leu Cys Val Arg Lys Arg Met Thr Leu 35 40 45 Gly Pro Gly LysVal Phe Ser Cys Asn Thr Ser Val Ile Thr Gln Ala 50 55 60 5 64 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 5 Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Lys ValGly Lys Ala Met 1 5 10 15 Tyr Ala Gln Arg Ile Ile His Ile Gly Pro GlyArg Pro Phe Ser Leu 20 25 30 Lys Pro Cys Val Lys Thr Pro Leu Cys Val ArgIle Arg Ile His Arg 35 40 45 Gly Tyr Gly Arg Ser Phe Ser Cys Asn Thr SerVal Ile Thr Gln Ala 50 55 60 6 64 PRT Artificial Sequence Description ofArtificial Sequence Human Immunodeficiency Virus 6 Arg Ile Lys Gln IleIle Asn Met Trp Gln Lys Val Gly Lys Ala Met 1 5 10 15 Tyr Ala Arg GlySer Ile His Leu His Pro Gly Arg Lys Phe Ser Leu 20 25 30 Lys Pro Cys ValLys Thr Pro Leu Cys Val Arg Lys Ser Ile Asn Met 35 40 45 Gly Pro Gly ArgAla Phe Ser Cys Asn Thr Ser Val Ile Thr Gln Ala 50 55 60 7 64 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 7 Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Lys ValGly Lys Ala Met 1 5 10 15 Tyr Ala Arg Lys Ser Ile Arg Ile Gly Pro GlyGln Thr Phe Ser Leu 20 25 30 Lys Pro Cys Val Lys Thr Pro Leu Cys Val ArgLys Ser Val Arg Ile 35 40 45 Gly Pro Gly Gln Thr Phe Ser Cys Asn Thr SerVal Ile Thr Gln Ala 50 55 60 8 64 PRT Artificial Sequence Description ofArtificial Sequence Human Immunodeficiency Virus 8 Arg Ile Lys Gln IleIle Asn Met Trp Gln Lys Val Gly Lys Ala Met 1 5 10 15 Tyr Ala Arg GluSer Ile Arg Ile Gly Pro Gly Gln Thr Phe Ser Leu 20 25 30 Lys Pro Cys ValLys Thr Pro Leu Cys Val Arg Arg Ser Ile Arg Ile 35 40 45 Gly Pro Gly GlnAla Phe Ser Cys Asn Thr Ser Val Ile Thr Gln Ala 50 55 60 9 64 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 9 Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Lys ValGly Lys Ala Met 1 5 10 15 Tyr Ala Arg Lys Gly Ile Arg Ile Gly Pro GlyGln Thr Phe Ser Leu 20 25 30 Lys Pro Cys Val Lys Thr Pro Leu Cys Val ArgLys Ser Ile Arg Ile 35 40 45 Gly Pro Gly Gln Ala Phe Ser Cys Asn Thr SerVal Ile Thr Gln Ala 50 55 60 10 64 PRT Artificial Sequence Descriptionof Artificial Sequence Human Immunodeficiency Virus 10 Arg Ile Lys GlnIle Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met 1 5 10 15 Tyr Ala ArgLys Ser Met Arg Ile Gly Pro Gly Gln Thr Phe Ser Leu 20 25 30 Lys Pro CysVal Lys Thr Pro Leu Cys Val Arg Lys Ser Ile Arg Ile 35 40 45 Gly Pro GlyGln Thr Leu Ser Cys Asn Thr Ser Val Ile Thr Gln Ala 50 55 60 11 64 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 11 Arg Ile Lys Gln Ile Ile Asn Met Trp Gln LysVal Gly Lys Ala Met 1 5 10 15 Tyr Ala Arg Lys Ser Val Arg Ile Gly ProGly Gln Thr Ser Ser Leu 20 25 30 Lys Pro Cys Val Lys Thr Pro Leu Cys ValArg Arg Ser Ile Arg Ile 35 40 45 Gly Pro Gly Gln Thr Phe Ser Cys Asn ThrSer Val Ile Thr Gln Ala 50 55 60 12 64 PRT Artificial SequenceDescription of Artificial Sequence Human Immunodeficiency Virus 12 ArgIle Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met 1 5 10 15Tyr Ala Arg Gln Ser Ile Arg Ile Gly Pro Gly Gln Ala Phe Ser Leu 20 25 30Lys Pro Cys Val Lys Thr Pro Leu Cys Val Arg Lys Ser Val Arg Ile 35 40 45Gly Pro Gly Gln Ala Phe Ser Cys Asn Thr Ser Val Ile Thr Gln Ala 50 55 6013 64 PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 13 Arg Ile Lys Gln Ile Ile Asn Met Trp Gln LysVal Gly Lys Ala Met 1 5 10 15 Tyr Ala Arg Lys Gly Ile His Ile Gly ProGly Gln Ala Phe Ser Leu 20 25 30 Lys Pro Cys Val Lys Thr Pro Leu Cys ValArg Lys Gly Ile Gly Ile 35 40 45 Gly Pro Gly Gln Thr Phe Ser Cys Asn ThrSer Val Ile Thr Gln Ala 50 55 60 14 64 PRT Artificial SequenceDescription of Artificial Sequence Human Immunodeficiency Virus 14 ArgIle Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met 1 5 10 15Tyr Ala Arg Glu Ser Ile Gly Ile Gly Pro Gly Gln Ala Phe Ser Leu 20 25 30Lys Pro Cys Val Lys Thr Pro Leu Cys Val Arg Lys Ser Met Arg Ile 35 40 45Gly Pro Gly Gln Thr Phe Ser Cys Asn Thr Ser Val Ile Thr Gln Ala 50 55 6015 46 PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 15 Arg Lys Ser Ile His Ile Gly Pro Gly Arg AlaPhe Ser Leu Lys Pro 1 5 10 15 Cys Val Lys Thr Pro Leu Cys Val Arg LysSer Ile His Ile Ala Pro 20 25 30 Gly Arg Ala Phe Ser Cys Asn Thr Ser ValIle Thr Gln Ala 35 40 45 16 52 PRT Artificial Sequence Description ofArtificial Sequence Human Immunodeficiency Virus 16 Arg Ile Lys Gln IleIle Asn Met Trp Gln Lys Val Gly Lys Ala Met 1 5 10 15 Tyr Ala Arg LysSer Ile His Ile Gly Pro Gly Arg Ala Phe Arg Lys 20 25 30 Ser Ile His IleAla Pro Gly Arg Ala Phe Ser Cys Asn Thr Ser Val 35 40 45 Ile Thr Gln Ala50 17 42 PRT Artificial Sequence Description of Artificial SequenceHuman Immunodeficiency Virus 17 Arg Ile Lys Gln Ile Ile Asn Met Trp GlnLys Val Gly Lys Ala Met 1 5 10 15 Tyr Ala Arg Lys Ser Ile His Ile GlyPro Gly Arg Ala Phe Arg Lys 20 25 30 Ser Ile His Ile Ala Pro Gly Arg AlaPhe 35 40 18 54 PRT Artificial Sequence Description of ArtificialSequence Human Immunodeficiency Virus 18 Arg Ile Lys Gln Ile Ile Asn MetTrp Gln Lys Val Gly Lys Ala Met 1 5 10 15 Tyr Ala Arg Lys Ser Ile HisIle Gly Pro Gly Arg Ala Phe Ser Leu 20 25 30 Lys Pro Cys Val Lys Thr ProLeu Cys Val Arg Lys Ser Ile His Ile 35 40 45 Ala Pro Gly Arg Ala Phe 5019 36 PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 19 Arg Lys Ser Ile His Ile Gly Pro Gly Arg AlaPhe Ser Leu Lys Pro 1 5 10 15 Cys Val Lys Thr Pro Leu Cys Val Arg LysSer Ile His Ile Ala Pro 20 25 30 Gly Arg Ala Phe 35 20 56 PRT ArtificialSequence Description of Artificial Sequence Human Immunodeficiency Virus20 Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met 1 510 15 Tyr Ala Ser Ile Gly Pro Gly Arg Ala Phe Ser Leu Lys Pro Cys Val 2025 30 Lys Thr Pro Leu Cys Val Ser Ile Gly Pro Gly Arg Ala Phe Ser Cys 3540 45 Asn Thr Ser Val Ile Thr Gln Ala 50 55 21 64 PRT ArtificialSequence Description of Artificial Sequence Human Immunodeficiency Virus21 Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met 1 510 15 Tyr Ala Arg Arg Asn Ile His Ile Gly Leu Gly Arg Arg Phe Ser Leu 2025 30 Lys Pro Cys Val Lys Thr Pro Leu Cys Val Arg Arg Ser Val Arg Ile 3540 45 Gly Pro Gly Gly Ala Met Ser Cys Asn Thr Ser Val Ile Thr Gln Ala 5055 60 22 64 PRT Artificial Sequence Description of Artificial SequenceHuman Immunodeficiency Virus 22 Arg Ile Lys Gln Ile Ile Asn Met Trp GlnLys Val Gly Lys Ala Met 1 5 10 15 Tyr Ala Arg Arg Ser Ile Pro Ile GlyPro Gly Arg Ala Phe Ser Leu 20 25 30 Lys Pro Cys Val Lys Thr Pro Leu CysVal Val Arg Lys Ile Pro Ile 35 40 45 Gly Pro Gly Ser Ser Phe Ser Cys AsnThr Ser Val Ile Thr Gln Ala 50 55 60 23 64 PRT Artificial SequenceDescription of Artificial Sequence Human Immunodeficiency Virus 23 ArgIle Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met 1 5 10 15Tyr Ala Arg Lys Arg Ile Pro Leu Gly Leu Gly Lys Ala Phe Ser Leu 20 25 30Lys Pro Cys Val Lys Thr Pro Leu Cys Val Arg Lys Gly Ile His Leu 35 40 45Gly Pro Gly Arg Ala Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala 50 55 6024 64 PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 24 Arg Ile Lys Gln Ile Ile Asn Met Trp Gln LysVal Gly Lys Ala Met 1 5 10 15 Tyr Ala Arg Lys Gly Ile His Met Gly ProGly Lys Ala Ile Ser Leu 20 25 30 Lys Pro Cys Val Lys Thr Pro Leu Cys ValArg Arg Gly Ile Pro Ile 35 40 45 Gly Pro Gly Arg Ala Phe Ser Cys Asn ThrSer Val Ile Thr Gln Ala 50 55 60 25 64 PRT Artificial SequenceDescription of Artificial Sequence Human Immunodeficiency Virus 25 ArgIle Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met 1 5 10 15Tyr Ala Arg Lys Ser Ile Arg Ile Gly Pro Gly Arg Ala Val Ser Leu 20 25 30Lys Pro Cys Val Lys Thr Pro Leu Cys Val Arg Arg Arg Ile Ser Ile 35 40 45Gly Pro Gly Arg Ala Phe Ser Cys Asn Thr Ser Val Ile Thr Gln Ala 50 55 6026 64 PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 26 Arg Ile Lys Gln Ile Ile Asn Met Trp Gln LysVal Gly Lys Ala Met 1 5 10 15 Tyr Ala Arg Arg Ser Ile His Leu Gly MetGly Arg Ala Leu Ser Leu 20 25 30 Lys Pro Cys Val Lys Thr Pro Leu Cys ValArg Arg Ser Ile His Met 35 40 45 Gly Leu Gly Arg Ala Phe Ser Cys Asn ThrSer Val Ile Thr Gln Ala 50 55 60 27 64 PRT Artificial SequenceDescription of Artificial Sequence Human Immunodeficiency Virus 27 ArgIle Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met 1 5 10 15Tyr Ala Arg Lys Gly Ile Asn Ile Gly Pro Gly Arg Ala Phe Ser Leu 20 25 30Lys Pro Cys Val Lys Thr Pro Leu Cys Val Arg Lys Gly Ile His Ile 35 40 45Gly Pro Gly Arg Thr Phe Ser Cys Asn Thr Ser Val Ile Thr Gln Ala 50 55 6028 64 PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 28 Arg Ile Lys Gln Ile Ile Asn Met Trp Gln LysVal Gly Lys Ala Met 1 5 10 15 Tyr Ala Ile Arg Ile Gly His Ile Gly ProGly Arg Ala Phe Ser Leu 20 25 30 Lys Pro Cys Val Lys Thr Pro Leu Cys ValArg Arg His Ile His Ile 35 40 45 Gly Pro Gly Arg Ala Phe Ser Cys Asn ThrSer Val Ile Thr Gln Ala 50 55 60 29 66 PRT Artificial SequenceDescription of Artificial Sequence Human Immunodeficiency Virus 29 ArgIle Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met 1 5 10 15Tyr Ala Arg Arg Lys Gly Ile His Ile Gly Pro Gly Arg Ala Ile Ser 20 25 30Leu Lys Pro Cys Val Lys Thr Pro Leu Cys Val Thr Gly Lys Ser Ile 35 40 45Arg Met Gly Leu Gly Arg Ala Trp Ser Cys Asn Thr Ser Val Ile Thr 50 55 60Gln Ala 65

What is claimed is:
 1. A peptide immunogen comprising, from theN-terminus to the C-terminus, beta sheet regions 20, 21, 2 and 3 of ahuman immunodeficiency virus (HIV) gp120 CCR5 binding site, wherein anHIV gp120 V3 loop sequence is present between said beta sheet regions 21and 2 and between said beta sheets regions 2 and
 3. 2. The peptideaccording to claim 1 wherein each of said V3 loop sequences comprisesfrom about 8 to about 16 amino acids.
 3. The peptide according to claim1 wherein said beta sheet regions correspond to disparate gp120 regions.4. A composition comprising at least two peptides according to claim 1.5. The composition according to claim 4 wherein said at least 2 peptidesare covalently linked.
 6. A method of inducing an immune response in apatient to HIV comprising administering to said patient at least onepeptide according to claim 1 in an amount and under conditions such thatsaid response is induced.
 7. A vaccine comprising a multiplicity ofpeptides according to claim 1 wherein said V3 loop sequences areselected so as to be representative of higher order structural motifspresent in a population of HIV isolates.
 8. The peptide according toclaim 1 wherein said peptide comprises a sequence selected from thegroup consisting ofRIKQIINMWQKVGKAMYA-RRSIPIGPGRAF-SLKPCVKTPLCV-VRKIPIGPGSSF-SCNTSVITQA;RIKQIINMWQKVGKAMYA-RKRIPLGLGKAF-SLKPCVKTPLCV-RKGIHLGPGRAI-SCNTSVITQA;RIKQIINMWQKVGKAMYA-RKGIHMGPGKAI-SLKPCVKTPLCV-RRGIPIGPGRAF-SCNTSVITQA;RIKQIINMWQKVGKAMYA-RKSIRIGPGRAV-SLKPCVKTPLCV-RRRISIGPGRAF-SCNTSVITQA;RIKQIINMWQKVGKAMYA-RRSIHLGMGRAL-SLKPCVKTPLCV-RRSIHMGLGRAF-SCNTSVITQA;RIKQIINMWQKVGKAMYA-RKGINIGPGRAF-SLKPCVKTPLCV-RKGIHIGPGRTF-SCNTSVITQA;RIKQIINMWQKVGKAMYA-IRIGHIGPGRAF-SLKPCVKTPLCV-RRHIHIGPGRAF-SCNTSVITQA;RIKQIINMWQKVGKAMYA-RRKGIHIGPGRAI-SLKPCVKTPLCV-TGKSIRMGLGRAW-SCNTSVITQA;RIKQIINMWQKVGKAMYA-RKSINIGPGRAF-SLKPCVKTPLCV-RKSIQIGPGRAF-SCNTSVITQA;RIKQIINMWQKVGKAMYA-RKSIHIGPGRAF-SLKPCVKTPLCV-RKSIHIAPGRAF-SCNTSVITQA;IKQIINMWQKVGKAMYA-RKSIHIGPGRAF-SLKPCVKTPLCV-RRRISMGPGRVL-SCNTSVITQA;RIKQIINMWQKVGKAMYA-RKRISLGPGRVY-SLKPCVKTPLCV-RKRMTLGPGKVF-SCNTSVITQA;RIKQIINMWQKVGKAMYA-QRIIHIGPGRPF-SLKPCVKTPLCV-RIRIHRGYGRSF-SCNTSVITQA;andRIKQIINMWQKVGKAMYA-RGSIHLHPGRKF-SLKPCVKTPLCV-RKSINMGPGRAF-SCNTSVITQA. 9.A composition comprising at least two of said peptides according toclaim
 8. 10. The peptide according to claim 1 wherein said peptidecomprises a sequence selected from the group consisting ofRIKQIINMWQKVGKAMYA-rksirigpGqtf-SLKPCVKTPLCV-rksVrigpGqtf-SCNTSVITQA;RIKQIINMWQKVGKAMYA-rEsirigpGqtf-SLKPCVKTPLCV-rRsirigpGqAf-SCNTSVITQA;RIKQIINMWQKVGKAMYA-rkGirigpGqtf-SLKPCVKTPLCV-rksirigpGqAf-SCNTSVITQA;RIKQIINMWQKVGKAMYA-rksMrigpGqtf-SLKPCVKTPLCV-rksirigpGqtL-SCNTSVITQA;RIKQIINMWQKVGKAMYA-rksVrigpGqtS-SLKPCVKTPLCV-rRsirigpGqtf-SCNTSVITQA;RIKQIINMWQKVGKAMYA-rQsirigpGqAf-SLKPCVKTPLCV-rksVrigpGqAf-SCNTSVITQA;RIKQTINMWQKVGKAMYA-rkGiHigpGqAf-SLKPCVKTPLCV-rkGiGigpGqtf-SCNTSVITQA;andRIKQIINMWQKVGKAMYA-rEsiGigpGqAf-SLKPCVKTPLCV-rksMrigpGqtf-SCNTSVITQA.11. A composition comprising at least two of said peptides according toclaim
 10. 12. A nucleic acid sequence encoding at least one peptideaccording to claim
 1. 13. A composition comprising at least one nucleicacid sequence encoding at least two of said peptides according toclaim
 1. 14. A method of inducing an immune response in a patient to HIVcomprising administering to said patient at least one nucleic acidsequence according to claim 12 under conditions such that said nucleicacid sequence is expressed, said at least one peptide is produced andsaid immune response is induced.